Retrotransposons, the important component of eukaryotic genome, are seeds of evolution and play great role in creating new genes. The compact Arabidopsis genome harbors over 200 Copia-like retrotransposons, but mostly silent. Here we isolated an expressed gene AtCopeg1 (Copia evolved gene 1), which shows higher than 90% identity to AtCopia95_I, the consensus sequence encoding AtCopia95 polyprotein. AtCopeg1 is the unique gene evolved from AtCopia95 family. It is an intron-containing gene with two alternative 3' ends. The transcript accumulation of AtCopeg1 is tissue-specific, also significantly affected by external hormones and abiotic stresses. The presence of regulatory elements in its promoter region (originating from AtCopia95_I and AtCopia95 long terminal repeat), is adequate for conferring its essential expression feature. Thus, AtCopeg1 is a versatile functional gene involved in many developmental and adaptive processes probably including the signaling crosstalk of hormone and nutrient stress. Our work highlighted the role of transposable elements in creating new functional genes, and will incite the enthusiasm for isolation and functional characterization of plant genes evolved from those previously considered as selfish and junk DNA.
BackgroundThe green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity.ResultsThe recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~ 60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~ 4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics.ConclusionsThe strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.
Background: The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. Results : The recombinant GFP was transiently expressed in an active form in agoinoculated N. benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~4 h and can recover 34.1 % of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics. Conclusions: The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP. Keywords: Green fluorescent protein, Plant virus, Transient gene expression, Aqueous two-phase system, Hydrophobic interaction chromatography
The maize genome remains abundant in molecular diversity, and the rich genetic diversity of maize starch-synthesis genes is crucial for controlling various grain traits. To explore the unique mechanism controlling the advantageous waxy trait and characterize the molecular feature of genes relevant to starch composition in two elite waxy inbreds, expression profiling combined with gene organization analysis was performed in them as compared to one normal inbred. Genotype-specific expression patterns were observed for most genes studied. The waxy inbreds were shown to contain mutations in multiple starch-synthesis genes, namely gbssI (wx), gbssIIb and isa2 (potentially isa3 too).The mis-splicing events directly accounted for wx loss of function. Contrarily, disruption of 5' and 3' transcript sequence may contribute to the absence of GbssIIb and Isa2 transcripts in waxy inbreds, respectively. Besides, the splicing of Sugary1 transcript was developmentally regulated in the normal inbred, and DNA polymorphisms were detected within SSIIIb-1 gene in waxy inbreds.
Plant-based expression platforms are currently gaining acceptance as a viable alternative for the production of recombinant proteins (RPs), but the degradation of RPs by proteases in cells hinders their superb potentials. Co-expression of a protease inhibitor (PI) shows promise as a strategy to prevent RP from proteolytic degradation in plants. However, competitive effects behind the PI-RP co-expression system may mask or obfuscate the in situ protective effects of a companion PI. Here, we explored the competitive effects by co-expressing reteplase (rPA) with three unrelated PIs, namely NbPR4, HsTIMP, and SlCYS8, in Nicotiana benthamiana leaves. Remarkably, the accumulation of rPA was significantly repressed by each of the three PIs, suggesting that the competitive effects may be common among the PIs. The repression can be attenuated by reducing the PI inoculum dose in the co-inoculation mixtures, showing a negative correlation between the PI abundance of the PI-RP system and competitive effects. Interestingly, when a replicating vector was used to modulate the relative abundance of PI and RP in vivo, rPA was still boosted even at the maximal testing dose of PI, indicating that the competitive effects reduced to an ignorable level by this in vivo approach. Furthermore, a 7- to 12-fold increase of rPA was achieved, proving that it is a useful way for stimulating the potentials of a companion PI by overcoming competitive effects. And, this approach can be applied to molecular farming for improving the RP yields of plant expression systems.
Background: The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. Results: The recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~4 h and can recover 34.1 % of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics. Conclusions: The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.
Background The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. Results The recombinant GFP was transiently expressed in an active form in agoinoculated N. benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~60% of total soluble proteins (TSPs). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no any changes in its spectroscopic characteristics. Conclusions The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.
Background: The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity. Results : The recombinant GFP was transiently expressed in an active form in agoinoculated N. benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~4 h and can recover 34.1 % of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics. Conclusions: The strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP. Keywords: Green fluorescent protein, Plant virus, Transient gene expression, Aqueous two-phase system, Hydrophobic interaction chromatography
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